All lanes : Anti-UBE3A antibody [EPR23077-14] (ab272168) at 1/1000 dilution

Lane 1 : HeLa (human cervix adenocarcinoma epithelial cell) treated with 10 uM MG-132 for 24 hours, whole cell lysate
Lane 2 : HEK-293T (human embryonic kidney epithelial cell) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Predicted band size: 100 kDa
Observed band size: 100,100 kDa

Blocking and diluting buffer and concentration: 5% NFDM/TBST.

Fresh lysate was used in lane 2. 

Exposure time: Lane 1: 3 minutes; Lane 2: 48 seconds.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa cells labelling UBE3A with ab272168 at 1/100 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing nuclear and weak cytoplasmic staining in HeLa cell line ab195889. Anti-alpha Tubulin antibody (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1000 dilution.

Indirect ELISA using ab272168 at varying antibody concentrations (1000-0 ng/ml) and Human UBE3A antigen at 1000 ng/ml. Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) at 1/2500 dilution was used as a secondary antibody.

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol-permeabilized HeLa (Human cervix adenocarcinoma epithelial cell) cells labelling UBE3A with ab272168 at 1/500 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

UBE3A was immunoprecipitated from 0.35 mg K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate with ab272168 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab272168 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate 10 ug

Lane 2: ab272168 IP in K-562 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab272168 in K-562 whole cell lysate

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 15 seconds

A 37 kDa degraded band is observed.

All lanes : Anti-UBE3A antibody [EPR23077-14] (ab272168) at 1/1000 dilution

Lane 1 : PC-12 (rat adrenal gland pheochromocytoma ) whole cell lysate
Lane 2 : PC-12 treated with 10 µM MG-132 for 4 hours, whole cell lysate
Lane 3 : RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) whole cell lysate
Lane 4 : RAW 264.7 treated with 10 µM MG-132 for 4 hours, whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 100 kDa
Observed band size: 100/37 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: 3 minutes.

Generation of the 37-kDa degraded fragment can be inhibited/reduced by MG-132 treatment (lanes 2 and 4).

All lanes : Anti-UBE3A antibody [EPR23077-14] (ab272168) at 1/1000 dilution

Lane 1 : Mouse spleen tissue lysate
Lane 2 : Rat brain tissue lysate
Lane 3 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 4 : K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate
Lane 5 : HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate
Lane 6 : HEK-293T (human embryonic kidney epithelial cell) whole cell lysate
Lane 7 : A549 (human lung carcinoma epithelial cell) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Predicted band size: 100 kDa
Observed band size: 100/37 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer and concentration: 5% NFDM/TBST.

A 37 kDa degraded band is observed. MG132 treatment or freshly made lysates can decrease the degradation. 

Exposure time: 3 minutes

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized RAW 264.7 cells labelling UBE3A with ab272168 at 1/100 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing nuclear and weak cytoplasmic staining in RAW 264.7 cell line. ab195889 Anti-alpha Tubulin antibody (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1000 dilution.

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol-permeabilized RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) cells labelling UBE3A with ab272168 at 1/50 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

UBE3A was immunoprecipitated from 0.35 mg RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) (treated with 10 μM MG-132 for 4 hours) whole cell lysate with ab272168 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab272168 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) treated with 10 μM MG-132 for 4 hours, whole cell lysate 10 ug

Lane 2: ab272168 IP in RAW 264.7 treated with 10 μM MG-132 for 4 hours, whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab272168 in RAW 264.7 treated with 10 μM MG-132 for 4 hours, whole cell lysate

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 10 seconds

A 37 kDa degraded band is observed.